Mycoplasma arthritidis T-cell mitogen

ABSTRACT

A method of purifying Mycoplasma arthritidis mitogen (MAM) to electrophoretic and sequence homogeneity is disclosed. A preparation of MAM purified according to this method was used to determine the sequence of the N-terminal 54 amino acids of MAM. A synthetic peptide consisting of amino acids 15-32 inhibited MAM-induced cell proliferation in vitro. The sequence of the N-terminal 54 amino acids was reverse translated, nucleotide probes were designed therefrom, and the MAM gene was selected from a genomic library. The MAM gene was sequenced and found to be contained on a 1107 bp DNA fragment. The primary translation product contains a 39 amino acid signal sequence and a 213 amino acid mature MAM (molecular weight 25,294). Amino acid sequence comparisons of MAM to bacterial and murine tumor virus superantigens showed regions of conservative sequence homology, including the region capable of inhibiting cell proliferation. Sequence homologies to HIV and other retrovirus proteins and to certain regulatory proteins were also detected. Strategies for blocking or immunizing against certain diseases, including autoimmune diseases, are disclosed.

This application is a divisional of application Ser. No. 08/165,038filed Dec. 10, 1993, now U.S. Pat. No. 5,639,869.

BACKGROUND OF THE INVENTION

This invention was made with government support under grants AI12103 andAM02255 awarded by the U.S. Department of Health and Human Services. Thegovernment has certain rights in the invention.

This invention relates to compositions and methods for preventing humandiseases including autoimmune diseases. More particularly, thisinvention relates to nucleic acid sequences and amino acid sequences ofMycoplasma arthritidis mitogen (MAM), synthetic oligonucleotides andpeptides having such sequences, a method of purifying MAM toelectrophoretic and sequence homogeneity, and methods of using suchsequences and purified MAM in preventing human disease.

In autoimmune disease, a breakdown of self-tolerance leads to generationof an immune response against a specific target antigen or antigens.Microbial agents have long been thought to trigger autoimmune diseasesby possessing antigenic determinants that are crossreactive withantigens on target organs. More recently, it has been suggested thatsuperantigens derived from bacteria, P. Marrack & J. Kappler, 248Science 705 (1990); B. Fleischer, 10 Immunol. Today 262 (1989),mycoplasma, B. Cole & C. Atkin, 12 Immunol. Today 271 (1991), orviruses, W. Frankel et al., 349 Nature 526 (1991); P. Dyson et al., 349Nature 531 (1991); Y. Choi et al., 350 Nature 203 (1991), may initiateautoimmune disease by activating specific anti-self T cell clones, J.White et al., 56 Cell 27 (1989); B. Cole et al., 144 J. Immunol. 425(1990), X. Paliard et al., 253 Science 325 (1991), or by forming asuperantigen bridge that crosslinks helper T (T_(H)) cells withpre-immune B cells, thereby causing polyclonal B cell activation andsecretion of autoimmune antibodies, S. Friedman et al., 34 ArthritisRheum. 468 (1991), W. Mourad et al., 170 J. Exp. Med. 2011 (1989). Infact, recent studies have shown that MAM can trigger, enhance, andexacerbate experimental autoimmune collagen-induced arthritis (CIA). B.Cole & M. Griffiths, 36 Arthritis Rheum. 994 (1993).

Superantigens are potent mitogens that activate T cells by a uniquepathway that binds the major histocompatibility complex (MHC) moleculeson accessory cell or B lymphocyte surfaces with specific β-chainvariable regions (V.sub.β) of the α/β T cell receptor for antigen (TCR)present on T cells. Thus, a particular superantigen may be recognized byvirtually all T cells that utilize a single or small group of TCRV.sub.β gene families. While there is some overlap, each superantigen isrecognized by its use of a distinct and characteristic set of TCRV.sub.β gene families. Further, superantigens bind selectively and withhigh affinity to class II MHC molecules. In the absence of antigenprocessing and in a non-MHC-restricted manner, superantigen-class II MHCantigen complexes on the antigen-presenting cell surface trigger theproliferation of T cells expressing the relevant TCR V.sub.β geneproducts. Finally, the in vivo presence of superantigens profoundlyalters the T cell repertoire. During the process of negative selectionwithin the thymus, a superantigen clonally eliminates thymocytes withTCR that bear V.sub.β gene products that recognize exactly thatsuperantigen. Superantigens include several staphylococcal enterotoxins,streptococcal pyrogenic exotoxins, a fragment of the group Astreptococcus M protein, murine self antigens such as the Mls loci geneproducts (now known to be encoded by murine tumor retroviruses) and anunknown B cell-specific antigen, and Mycoplasma arthritidis T cellmitogen (MAM).

Mycoplasmas are the smallest self-replicating prokaryotes and areparasites of humans, birds, insects, plants, and virtually all otherhigher life forms. Mycoplasmas are the most common cause ofnaturally-occurring acute and chronic arthritis in many animal species.M. arthritidis is a naturally-occurring arthritogen of rodents thatcauses a chronic, relapsing disease that, histologically, closelyresembles human rheumatoid arthritis. MAM was discovered when liveorganisms and culture supernatants of M. arthritidis were shown toinduce the proliferation of, and elicit the differentiation of,cytolytic cells in mouse splenocytes. B. Cole et al., 127 J. Immunol.1931 (1981). An insoluble, presumably membrane-bound B-cell mitogen wasfound to be associated with mycoplasma cells and was stable at 100° C.In contrast, a soluble T-cell mitogen was present in culturesupernatants and was heat labile at 56° C. This heat labile T-cellmitogen is MAM. MAM was then shown to be a potent T-cell mitogen andinducer of gamma-interferon (IFN-γ) for both murine and humanlymphocytes. B. Cole et al., 128 J. Immunol. 2013 (1982); B. Cole & R.Thorpe, 131 J. Immunol. 2392 (1983); B. Cole & R. Thorpe, 43 Infect.Immun. 302 (1984); T. Moritz et al., 20 Scand. J. Immunol. 365 (1984);H. Kirchner et al., 20 Scand. J. Immunol. 133 (1984); H. Kirchner etal., 4 J. Interferon Res. 389 (1984).

MAM is produced to maximal titer in senescent broth cultures of M.arthritidis. Purification is difficult because MAM is produced in smallamounts, is heat and acid (pH<7.0) labile, and has great affinity forsurfaces and large molecules, especially nucleic acids. Gel filtrationof culture supernatants, at an ionic strength of about 0.5M, indicatedthat MAM has a molecular mass of about 15 kD whereas PAGE suggested themolecule was about 30 kD. C. Atkin et al., 137 J. Immunol. 1581 (1986);H. Kirchner et al., 24 Scand. J. Immunol. 245 (1986). Although Kirchneret al. claimed partial purification of MAM, their purification stepswould have yielded ≦200-fold purification in the best of hands. Sincetheir mitogenic assay was merely qualitative, they were unable to showyield or specific activity (mitogenicity per unit. protein). J. Homfeldet al., 7 Autoimmunity 317 (1990), have also described partialpurification of MAM. Using a quantitative assay for MAM, C. Atkin etal., 137 J. Immunol. 1581 (1986), to achieve 200,000-fold purification.The calculated purification of MAM by the final gel filtration stepimplies measurement of protein, but the method was not given nor was aprofile of protein or absorbance shown. Active fractions corresponded tothe elution volumes of 15-20 kD standards, but no stainable protein bySDS-PAGE was identified nor was an amino acid sequence reported.

One of the major activities of MAM is its ability to cause aproliferation of lymphocytes from certain strains of mice, but not ofothers. Lymphocytes from BALB/c and C3H mice are readily activatedwhereas those of C57BL/10 mice fail to undergo proliferation in responseto exposure to MAM. This negative or weak response of C57BL/10 miceenabled mapping one of the genes which control MAM reactivity to the I-Eregion of the murine H-2 MHC. Dependence upon MHC-bearing accessorycells for MAM-induced T-cell proliferation was consistent with thisconclusion. This specificity for I-E bearing cells suggested that theI-E molecule might be the binding site for MAM. The fact that onlysplenocytes from I-E-bearing mouse strains could remove MAM activityfrom solution and liposomes with incorporated I-E, but not with I-A,molecules could present MAM to T cells supported this hypothesis. Thereis substantial evidence that the conserved a chain of the I-E molecule,or a combinatorial determinant between E.sub.α and other β chains, bearsthe MAM receptor. Evidence of this includes ATFR5 mice which lackE.sub.β respond to MAM through combinatorial E.sub.α A.sub.β molecules,antibodies to a monoclonal antibody specific for E.sub.α totally blockMAM-induced proliferation, E.sub.α transgenic mice on a C57BL/10background present MAM, and transfected fibroblasts expressing E.sub.αE.sub.β or E.sub.α A.sub.β present MAM, whereas fibroblasts expressingA.sub.α A.sub.β do not. B. Cole et al., 127 J. Immunol. 1931 (1981); B.Cole et al., 128 J. Immunol. 2013 (1982); B. Cole et al., 129 J.Immunol. 1352 (1982); B. Cole et al., 136 J. Immunol. 3572 (1986); M.Bekoff et al., 139 J. Immunol. 3189 (1987); M. Matthes et al., 18 Eur.J. Immunol. 1733 (1988); B. Cole et al., 144 J. Immunol. 420 (1990).

MAM, like other superantigens, is recognized by V.sub.β chain segmentsof the α/β TCR. This was demonstrated in progeny of test-crosses betweenRIIIS mice, which have massive deletions in their V.sub.β α/β T-cellrepertoire, with (RIIIS×B10.RIII)F1 hybrids. B10.RIII mice contains allV.sub.β genes. Reactivity of lymphocytes with MAM cosegregated withexpression of V.sub.β 8-bearing cells. Thus, at least the V.sub.β 8 TCRgene family was involved in recognition of MAM. In other experiments,clonal expansion of MAM-reactive BALB/c cells in vitro showed theactivated cells expressed V.sub.β 8.1, V.sub.β 8.2, V.sub.β 8.3, andV.sub.β 6. MAM expansion of C57BR lymphocytes, which lack the V.sub.β 8genes, resulted in strong expression of V.sub.β 6 in the activatedpopulation. Similarly, it has been shown that MAM can use TCRsexpressing V.sub.β 5.1. These specificities of MAM for certain TCR geneswas reported in B. Cole et al., 144 J. Immunol. 425 (1990); L. Baccalaet al., 35 Arthritis Rheum. 434 (1992). In rats, MAM-reactive V.sub.βare homologous to the MAM-reactive V.sub.β of mice, with one exception.Engagement of rat V.sub.β 5.1, V.sub.β 6, V.sub.β 8.1, and V.sub.β 8.2,but not V.sub.β 8.3 were observed. In humans, the engaged V.sub.βincluded primarily V.sub.β 19.1 (alternatively termed V.sub.β 17.1) and,to a lesser extent, V.sub.β 3.1, V.sub.β 11.1, V.sub.β 12.1, and V.sub.β13.1. R. Baccala et al., 35 Arthritis Rheum. 434 (1992). More recentexperiments have shown that both genomic composition and allelicpolymorphisms at the V.sub.β chain segment of the TCR exert profoundeffects upon the pattern of V.sub.β that are used by MAM. Thus, inV.sub.β^(b) haplotype mice, without genomic deletions of V.sub.β genes,V.sub.β 5.1, 6, 8.1, 8.2, and 8.3 engage MAM. In V.sub.β^(a) mice, withdeletions in V.sub.β 5.1 to 5.3, 8.1 to 8.3, 9, 11, 12, and 13, therewas significant expansion of V.sub.β 6-expressing cells and lesserexpansions of V.sub.β 1-, 7-, and 16-expressing cells. In V.sub.β^(c)mice, with deletions of the same V.sub.β genes deleted in V.sub.β^(a)and additional deletions in V.sub.β 6, 15, and 17, there was a dominantexpansion of V.sub.β 7 and V.sub.β 1, and a slight expansion of V.sub.β3.1-expressing cells. B. Cole et al., 150 J. Immunol. 3291 (1993). Usageof V.sub.β 8 gene products is fairly common among other microbialsuperantigens, however V.sub.β 6 is only used by MAM and the Mls 1^(a)antigen now known to be a murine retroviral superantigen.

MAM can also activate human peripheral blood lymphocytes; this reactiontoo is dependent upon MHC molecules. The human MHC HLA-DR molecule, theequivalent of the murine H2 I-E molecule, appears to possess the MAMbinding site. Evidence of this includes inhibition of T-cellproliferation by anti-HLA-DR antibodies, production of IFN-γ andinduction of cytolytic cells in response to MAM, and presentation of MAMto human T-cells by cells transfected with I-E and the inhibition of theresponse by anti-I-E monoclonal antibodies. MAM can produceproliferation of human T-cells regardless of whether the cells expressCD4 or CD8 molecules. TCR α/β-negative, γ/δ-positive cells also respondto MAM in the presence of appropriate accessory cells. R. Daynes et al.,129 J. Immunol. 936 (1982); B. Cole & R. Thorpe, 131 J. Immunol. 2392(1983); M. Matthes et al., 18 Eur. J. Immunol. 1733 (1988); R. Baccalaet al., 35 Arthritis Rheum. 434 (1992).

The response of human cells to MAM has always been found to be weakerthan that of mouse cells and weaker than to lectin mitogens. In a directcomparison, human cells responded better to staphylococcal superantigensthan to MAM, and mouse cells responded better to MAM. B. Fleischer etal., 146 J. Immunol. 11 (1991). This difference seems to issue fromdifferences in the MHC/superantigen interaction since lymphocytes fromtransgenic mice expressing human MHC molecules respond better tostaphylococcal superantigens than to MAM.

The apparent ability of individual superantigen molecules to interactsimultaneously with MHC molecules on accessory cells and B cells, aswell as with V.sub.β TCRs on T-cells, raises the possibility thatsuperantigens might be able to initiate a B-T_(H) cell collaborationresulting in polyclonal B cell activation. Peripheral blood lymphocytesfrom normal individuals or rheumatoid arthritis patients secretedsignificantly higher levels of IgG when co-cultured in vitro with MAMand pokeweed mitogen. Further, purified B cell cultures or B cellsincubated with MAM-reactive T_(H) cells failed to secrete significantlevels of IgM. However, when B cells were briefly exposed to MAM or whenMAM was added to B-T_(H) cell mixtures, high levels of IgM wereproduced. This is important because abnormal B-T_(H) cell interactionsmimic the interaction seen in graft versus host disease that has beenused as a model of systemic lupus erythematosus (SLE). In SLE, abnormalB cell reactivity results in production of a wide range ofautoantibodies. P. Emery et al., 12 J. Rheumatol. 217 (1985); J. Tumanget al., 171 J. Exp. Med. 2153 (1990); S. Friedman et al., 34 ArthritisRheum. 468 (1991).

M. arthritidis also causes a severe suppurative arthritis in rats whichcan also be associated with uveitis, C. Thirkill & D. Gregerson, 36Infect. Immun. 775 (1982), conjunctivitis, urethritis, lethargy, andparalysis, J. Ward & R. Jones, 5 Arthritis Rheum. 163 (1962). MAM canactivate rat lymphocytes. B. Cole et al., 36 Infect. Immun. 662 (1982).Splenic cells from inbred rat strains August, Buffalo, DA, Lewis, WistarFurth, and (LEW x BN)F1 all responded well to MAM, but BN and MAXX ratsresponded very weakly or not at all. Genetic analysis showed thatnon-RT1 genes control responsiveness to MAM. Both responder andnonresponder splenic cells could bind MAM. These results contrast withthe results obtained with non-responder mouse strains, wherein the cellsfailed to bind MAM due to the absence of the E.sub.α chain of the I-Emolecule. B. Cole et al., 129 J. Immunol. 1352 (1982); B. Cole et al.,136 J. Immunol. 2364 (1986).

Interestingly, the genetics of MAM-induced activation of rat lymphocytesresembles that of susceptibility to two experimentally-inducedautoimmune diseases, experimental allergic encephalomyelitis (EAE) andcollagen-induced arthritis (CIA). Thus, (LEW x BN)F1 rats aresusceptible to both EAE and CIA, and responsiveness to MAM is a dominanttrait, whereas (DA x BN)F1 rats are resistant to both EAE and CIA, andresponsiveness to MAM is recessive. In both EAE and CIA, T-cellsexpressing V.sub.β TCRs are involved in disease pathogenesis. Since ratand mouse V.sub.β TCRs are quite similar, it is not surprising that MAMalso activates rat V.sub.β 8-bearing T-cells. L. Baccala et al., 35Arthritis Rheum. 434 (1992).

Importantly, this similarity between the genetic predisposition to CIAand lymphocyte reactivity to MAM is now known to be due to involvementof similar V.sub.β chain segments of the TCR on T cell surfaces. Thus, Tcells bearing V.sub.β 6, V.sub.β 7. and V.sub.β 8 participate in CIA. T.Haqqi et al., 89 Proc. Nat'l Acad. Sci USA 1253 (1992). These sameV.sub.β TCRs are also activated by MAM, B. Cole et al., 150 J. Immunol.3291 (1993), thus presenting a mechanism whereby superantigens mightactivate autoimmune disease. In fact, recent studies, B. Cole & M.Griffiths, 36 Arthritis Rheum. 944 (1993), have demonstrated that theintravenous injection of MAM (1) into mice suboptimally immunized withcollagen causes a triggering of arthritis, (2) into mice convalescingfrom CIA results in a flare of disease activity, and (3) into mice justafter injection of collagen causes an acceleration of the development ofarthritis.

MAM is also thought to play a role in the pathogenicity of M.arthritidis by causing immunosuppression of the host. M. arthritidis isfrequently harbored in the respiratory tract of apparently healthy miceand rats. Its presence may be undetectable without extensive culturingsince an antibody response may not be present. M. Davidson et al., 8Curr. Microb. 205 (1983). Even in experimentally-injected mice and rats,where complement-fixing antibodies are rapidly produced, the immuneresponse to M. arthritidis is defective. Neutralizing orgrowth-inhibiting antibodies, which play a major role in the control ofmycoplasma infections, are not produced against M. arthritidis inrodents. Opsonizing antibodies are likewise not produced. Probably forthese reasons, mycoplasmemia persists for up to 3 weeks in theperipheral circulation of intravenously-injected animals. B. Cole etal., 98 J. Bacteriol. 930 (1969); B. Cole & J. Ward, 7 Infect. Immun.691 (1973); B. Cole & J. Ward, 8 Infect. Immun. 199 (1973).

MAM may be responsible for depressed host defenses. Mycoplasmas arecleared faster from the peripheral circulation of mouse strains whichlack functional I-E molecules than from strains possessing I-E. B. Coleet al., 41 Infect. Immun. 1010 (1983). Lymphocytes taken fromI-E-bearing mice injected intravenously with MAM exhibit an impairedability to proliferate in response to MAM, and, to a lesser extent, tolectin mitogens. B. Cole & D. Wells, 58 Infect. Immun. 228 (1990). MAMalso appears to suppress other T-cell functions, such as contactsensitivity to dinitrofluorobenzene (DNFB) and can prolong skin graftsacross H-2 and non-H-2 barriers. In contrast, MAM appears not to haveany consistent suppressive effect in vivo on B-cell functions, but,instead, enhances B-cell activity.

MAM also appears at least partially responsible for reactions involvingtoxicity and necrosis in experimentally-injected mice. One of theearliest symptoms following intravenous injection of large numbers of M.arthritidis is a toxic shock syndrome that is analogous to the humancondition caused by a staphylococcal superantigen. Symptoms includelethargy, ruffled fur, conjunctivitis, fecal impaction, and death insome individuals. These effects were H-2 restricted in that animals withMAM-reactive lymphocytes were susceptible, whereas animals withMAM-nonreactive lymphocytes were resistant. In part, this reaction maybe due to liberation of lymphokines and other inflammatory moleculesmediated by MAM-induced activation of lymphocytes and macrophages sincelarge doses of highly purified MAM yielded a similar toxic syndrome, butof much lesser duration and severity. B. Cole et al., 41 Infect. Immun.1010 (1983); B. Cole & D. Wells, 58 Infect. Immun. 228 (1990).

MAM also appears to play a role in dermal necrosis induced bysubcutaneous injection of M. arthritidis in susceptible animals.Susceptible mice possess functional I-E, whereas mice lacking functionalI-E developed a suppurative abscess but without dermal damage. B. Coleet al., 85 J. Invest. Dermatol. 357 (1985).

OBJECTS AND SUMMARY OF THE INVENTION

An object of the present invention is to provide MAM that is purified tohomogeneity.

Another object of the invention is to provide a method of purifying MAMto homogeneity.

A further object of the invention is to provide a purifiedoligonucleotide containing a nucleotide sequence encoding MAM.

Yet another object of the invention is to provide an array ofoligopeptides that either mimic or inhibit various biological activitiesof MAM.

A further objective of the invention is to provide homogeneous MAM orspecific MAM peptides for use as reagents to modify immune reactivity invitro or in vivo and to establish model systems to study the mechanismsof development of autoimmune disease.

Still another object of the invention is to provide a method of blockingflares of human diseases that are caused by exposure to superantigens.

Another object of the invention is to provide a method of immunizingagainst human diseases that are caused, triggered by, or made moresevere by exposure to superantigens.

These and other objects may be accomplished by providing a method ofpurifying MAM to electrophoretic and sequence homogeneity from senescentcultures of M. arthritidis by recovering culture components that aresoluble in 50% saturated (NH₄)₂ SO₄ and insoluble in 80% saturated(NH₄)₂ SO₄. These culture components are then back extracted in 1M(NH₄)₂ SO₄ ; insoluble residues are sedimented and discarded whilesoluble components are retained. These soluble components are subjectedto fractionation by gel filtration in very high ionic strength, slightlyalkaline buffer, and fractions containing proteins of approximatemolecular weight of 30,000 are recovered. These fractions are pooled,then the buffer of the pooled fractions is changed to low conductivityneutral buffer. Pool components that are retained on a cation exchangecolumn after a starting buffer wash are eluted as a single 280nm-absorbing fraction by frontal elution with neutral high salt buffer.The buffer of this recovered fraction is again changed to lowconductivity neutral buffer; the many protein components of the singlefraction are then fractionated by linear gradient elution with a highsalt buffer on an FPLC (the proprietary "Fast Protein LiquidChromatography" system of Pharmacia-LKB Biotechnology, Inc., Piscataway,N.J.) cation exchange chromatographic column. Active fractions, asdetermined by murine lymphocyte proliferation assay, are placed in highmolarity salt buffer and subjected to FPLC hydrophobic interactionchromatography with reverse gradient elution to low salt. Elution ofproteins simultaneously monitored at 214 and 280 nm. The minuscule,last-eluting peak is recovered in one or two small fractions, and ishomogeneous MAM.

Another aspect of the invention is providing homogeneous MAM proteinpurified by the method summarized above. Only by having homogeneous MAMpreparations can sequence information be obtained. Such sequence wasdetermined for the N-terminal 54 amino acids of MAM by direct sequencingby the Edman method, yielding the amino acid sequence listed as SEQ IDNO:1.

Another aspect of the invention is providing a purified oligonucleotidehaving a nucleotide sequence encoding MAM. The MAM gene was selectedfrom a phage library of clones that spanned the entire genome of M.arthritidis PG6. This was achieved by first designing an oligonucleotideprobe, SEQ ID NO:2, from a reverse translation of SEQ ID NO:1. Thisprobe was used to select clones containing parts or all of the MAM gene,which clones were ordered and sequenced to yield the complete nucleotidesequence of the MAM gene, SEQ ID NO:3. It was thus determined that theMAM gene encodes a 39 amino acid signal or pre-peptide and a 213 aminoacid mature MAM, SEQ ID NO:4.

Another aspect of the invention is providing an oligopeptide thatinhibits MAM-induced cell proliferation. Three synthetic peptides, SEQID NO:5, SEQ ID NO:6, and SEQ ID NO:7, corresponding to amino acids1-13, 27-44, and 15-32 of mature MAM were made. In competitiveinhibition assays in vitro, the peptide having the sequence disclosed inSEQ ID NO:7 inhibited MAM-induced cell proliferation while SEQ ID NO:5and SEQ ID NO:6 did not.

Another aspect of the invention is providing therapeutic regimens forthe treatment of human diseases that are caused, triggered, orexacerbated by exposure to superantigens. Superantigens have beenpostulated to play roles in the development of human rheumatic diseasesas well as of AIDS. Treatments may include immunization of individualsagainst a superantigen or derivative peptide, or injection of homologouspeptides to directly block action of the superantigen. Amino acidsequence comparisons of MAM to bacterial superantigens, murine tumorvirus superantigens, HIV and other retrovirus superantigens, and certainregulatory proteins showed small regions of conservative sequencehomology. These regions were not co-linear as regards N-to-C-terminalalignment of the proteins and thus could not represent evolutionarilydivergent protein-protein homologies; these regions were on the contraryconsidered as "potential shared epitopes" that might arise byevolutionary convergence of fundamentally different proteins. One such"potential shared epitope" was within the sequence of the oligopeptideidentified as SEQ ID NO:7 and which inhibited MAM-induced cellproliferation in vitro. Administering effective doses of oligopeptidescontaining conservative homologous regions will permit blocking ofsuperantigen disease-causing activity or immunization against thesuperantigens to prevent human diseases caused by exposure tosuperantigens. Similarly, the ability to block or mimic the variousbiological activities of MAM in animals and tissue culture would be oftremendous experimental utility.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an elution profile from the second cation exchangechromatography step of MAM purification on Mono-S Sepharose and thespecific activity of the fractions.

FIG. 2 shows an elution profile from the hydrophobic interactionchromatography step of MAM purification on alkyl-Superose and thespecific activity of the fractions.

FIG. 3 shows an SDS-gradient polyacrylamide gel showing the homogeneityof MAM.

FIG. 4 shows the effects of synthetic peptides, SEQ ID NO:5, SEQ IDNO:6, and SEQ ID NO:7, on MAM-induced lymphocyte proliferation.

FIG. 5 shows small regions of the primary sequence of MAM that resembleregions of Gram-positive bacterial and retroviral superantigens.

FIG. 6 shows sequence similarities of Gram-positive bacterialsuperantigens to potential epitopic region 1 of MAM.

FIG. 7 shows sequence similarities of retroviral superantigens and HIV-1proteins to potential epitopic region 1 of MAM.

DETAILED DESCRIPTION OF THE INVENTION

Purification to Homogeneity of MAM

Senescent cultures of various strains of M. arthritidis, such as14124p10 and PG-6, contain MAM as a soluble, extracellular protein. MAMin typical cultures has great affinity for, and is thus difficult orimpossible to separate from nucleic acids and other solublemacromolecules that are present in the classic Edward-Hayflickmycoplasma medium and its like. However, homogeneous preparations of MAMwere obtained by the following procedure.

M. arthritidis PG-6 was obtained from the American Type CultureCollection (Rockville, Md.; accession number 19611). Strain PG-6produces higher levels of MAM than most other strains that have beentested. Inocula consisted of deep-frozen 1 ml aliquots of exponentiallygrowing M. arthritidis PG-6. PG-6 was grown under microaerophiliccondition in very gently swirled "boiled medium" (2 liters in each oftwo 4-6 liter Erlenmeyer flasks) at 37° C.

"Boiled medium" is an optically clear supernatant of autoclavedEdward-Hayflick medium, L. Hayflick, 23 Tex. Rep. Biol. Med. Supp.(1)285 (1965), amended withN-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES),arginine.HCl, and penicillin G, C. Atkin et al., 137 J. Immunol. 1581(1986). A batch of 4 l finished medium is made as follows. The followingingredients are mixed together in a 10 l stainless steel beaker: 4250 mlultrapure water; 750 ml non-heat-treated horse serum (HyClone Labs,Logan, Utah) (final concentration, 15% v/v); 84 g "Bacto" PPLO Brothwithout Crystal Violet (Difco Lab., Detroit, Mich.) (finalconcentration, 2.1% w/v); and 62.5 g Fleischmann's dry baker's yeast(final concentration of 1.25% w/v). The beaker of mixture is thencovered with aluminum foil and autoclaved for 40 minutes at 15 psi (132°C.). With occasional and very gentle stirring, the beaker of nowcustard-like mixture is chilled in a well-stirred ice bath, then left tostand overnight on ice. The mixture is then centrifuged at 4° C. for 30minutes at about 11,000 relative centrifugal force (RCF) withoutbraking. The essentially optically clear supernatant solution (4 l) isdecanted through cheesecloth sheets and the residue is discarded.Manipulations to this point preclude maintenance of asepsis; thesesteps, however, effect denaturation, precipitation, and removal of mostinterfering macromolecules. Next, HEPES and arginine.HCl are added to1.0% (w/v) and 0.5% (w/v), respectively, and the solution is thentitrated to pH 7.0 by dropwise addition of ≦5 ml concentrated aqueousNaOH. The solution is then sterilized by autoclaving for 15 minutes at132° C. and 15 psi. After it has cooled, penicillin G is added to 1000U/ml. Attaining sufficient optical clarity of the medium may requiresupplementation or replacement of the second autoclaving step bypressure aseptic ultrafiltration on large-diameter 0.22 μm MF filters(Millipore Corp., Bedford, Mass.).

Growth of the organisms is monitored turbidimetrically until a maximum(A₆₀₀ ≈0.2/cm) is reached in about 52 hours, then purification steps areperformed as rapidly as possible. The whole warm culture is measured forvolume, and is then brought to 50% saturation with respect to (NH₄)₂ SO₄by addition and dissolution of crushed high-purity (NH₄)₂ SO₄ (313 g/laccording to saturation tables in A. Green & W. Hughes, 1 MethodsEnzymol. 67 (1955)). The pH is then adjusted to 7-8 by dropwisetitration with concentrated aqueous ammonia. The mixture is thencentrifuged at 4° C. for 30 minutes at about 11,000 RCF without braking.The supernatant is recovered and the pellet of salted-out components isdiscarded. The measured volume of supernatant is similarly brought to800% saturation with respect to (NH₄)₂ SO₄, the pH adjusted as describedabove, and then the mixture is centrifuged as before. This time thepellet is saved and the supernatant is discarded. The pellet isback-extracted by addition of water to a total volume of 60 ml(calculated to give about a 1M (NH₄)₂ SO₄ solution of appropriate volumefor the first chromatographic step). Insoluble residue is thensedimented and discarded.

The MAM-containing extract solution is then subjected to gel filtrationchromatography on a 2 liter column (diameter:height=1:5-10) of mediumgrade Sephadex G-50 (Pharmacia-LKB) equilibrated with a very high ionicstrength, slightly alkaline buffer, such as 10 mM Tris-HCl, pH 8.3, 1M(NH₄)₂ SO₄ (Buffer 1). Medium grade Sephadex G-50 is composed of dextrancross linked with epichlorohydrin as beads with wet diameters of 100-300μm, with porosity adjusted for resolution of globular proteins ofnominal molecular weights of 1,500 to 30,000. Saved and pooled were theMAM-containing fractions at elution volumes previously determined forcarbonic anhydrase 30-kDalton molecular weight standard. Conductivityand pH of the pool were decreased to the values of Buffer 2 (10 mMpotassium phosphate, pH 7.2) by dialysis or by repeated dilution withBuffer 2 and centrifugal ultrafiltration andconcentration-ultrafiltration in Centriprep-10 tubes (Amicon, Danvers,Mass.).

The MAM-containing fraction is then adsorbed onto a 30 ml(diameter:length=1:5-10) column of Fast Flow Sepharose-S cation exchangeresin (a sulfonated and proprietary cross linked agarose asmean-diameter 90 μm beads; Pharmacia-LKB) that had already beenequilibrated with Buffer 2. The column is then washed with more Buffer 2until A₂₈₀ of the effluent returns to baseline. Frontal elution of thestill very impure active factor is achieved with a high salt buffer,such as 0.5M potassium phosphate, pH 7.2 (Buffer 3). One peak of 280nm-absorbing material is eluted by this step and is saved. Electrolytesof the MAM-containing fraction are equilibrated to those of Buffer 2 asbefore.

A second cation exchange chromatography step is then performed byadsorbing the MAM-containing fraction onto an HR 5/10 FPLC column (1 mlbed volume) of Mono-S cation exchange resin (a sulfonated proprietaryhydrophilic resin as monodisperse 10 μm beads; Pharmacia-LKB),pre-equilibrated with Buffer 2. The column is washed with Buffer 2 untilA₂₈₀ returns to baseline. The active factor is eluted in one or two 0.5ml fractions amid the next 20 ml of column flow with a linear gradientto high salt, such as 40% v/v Buffer 3. Fractions (0.5 ml) are collecteddirectly into Centricon-10 (Amicon) centrifugal ultrafiltrationconcentrator tubes. Fractions are selected by murine lymphocyteproliferation assay (MLPA) or using the 280-nm elution profile (FIG. 1)and assays of similar, previous runs as a guide. FIG. 1 shows elution ofMAM from a Mono-S Sepharose column. The elution time and volumecommenced with the start of the salt gradient. "Salt" indicates theprogrammed molarity of eluant potassium phosphate, pH 7.2. Saltconcentration in simultaneously eluting solution would be shown by thesame gradient shifted approximately 2 ml (or 2 min.) to the right. Theprofile of eluted protein is given by the A₂₈₀ /cm curve. Specificmitogenic activity (S.A.) of 0.5-ml fractions is shown in terms of(U/ml)÷(A₂₈₀ /cm) or, approximately, U/(mg protein). The bulk ofmitogenic activity eluted in fractions 6-8 in this example.Chromatographic runs vary sufficiently that fractions must usually beassayed for mitogen rather than simply chosen from the A₂₈₀ profile.

Selected fractions from the second cation exchange are typically about10% pure MAM and suffice for many biological experiments, especiallyafter ultrafiltration to remove high salt and addition of stabilizingcarrier proteins such as pyrogen- and mitogen-free bovine serum albumin,or murine or human serum.

For the final purification step of obtaining homogeneous MAM, selectedfractions are not manipulated except to add solid (NH₄)₂ SO₄ to bringthe salt concentration to 2 molar. These fractions are applied asrapidly as possible to an HR 5/10 FPLC column (1 ml bed volume) ofAlkyl-Superose hydrophobic interaction resin (a proprietarily crosslinked, proprietarily neopentyl-derivatized agarose as monodisperse 10μm beads; Pharmacia-LKB) with starting conditions of 2M (NH₄)₂ SO₄, 50mM potassium phosphate, pH 7.2 (Buffer 4). After washing with thisbuffer until the A₂₈₀ returns to baseline, a linear reverse gradient ismade over the next 20 ml of eluant to 50 mM potassium phosphate, pH 7.2(Buffer 5). Simultaneous monitoring of the effluent at 214 and 280 nmshows several bands of eluted contaminant proteins that are followed bya final, discrete and weak absorbance band (FIG. 2) that typicallycontains about 25 μg of homogeneous MAM (see next paragraph) in about 1ml of 50 mM potassium phosphate with roughly 1.4M (NH₄)₂ SO₄, pH 7.2 .FIG. 2 shows elution of MAM from an alkyl Superose column for the sameMAM preparation shown in FIG. 1. The dimensions of the figure are thesame as for FIG. 1, except "Salt" indicates the molarity (throughout a2M to 0M gradient) of (NH₄)₂ SO₄ in 50 mM potassium phosphate buffer, pH7.2. Table 1 characterizes the purification scheme by overall changeswrought on MAM-containing fractions. Most surfaces will adsorb ordenature purified MAM while glass will destroy it. Thus, the activefractions should be collected directly into Centricon-10 (Amicon) tubesto allow subsequent removal of salt or change of buffer.

                                      TABLE 1    __________________________________________________________________________    Purification of extracellular MAM.                               Specific                           Mitogen                               Mitogenic                                    Net Fold              Volume                  Protein                      Mitogen                           Yield                               Activity                                    Purifica-    Fraction  (ml)                  (mg/ml)                      (U/ml)                           (%) (U/mg)                                    tion    __________________________________________________________________________    Preparation No. 69    Whole     4000                  17.2                      2.1 × 10.sup.3                           100 1.2 × 10.sup.2                                    1    culture.sup.a    Alkyl     0.5 0.015                      0.2 × 10.sup.6                           1   0.1 × 10.sup.8                                    0.1 × 10.sup.6    Superose    12    13        0.5 0.030                      1.1 × 10.sup.6                           7   0.4 × 10.sup.8                                    0.3 × 10.sup.6    14        0.5 0.020                      1.9 × 10.sup.6                           12  1.0 × 10.sup.8                                    0.8 × 10.sup.6    15        0.5 0.010                      0.1 × 10.sup.6                           1   0.1 × 10.sup.8                                    0.1 × 10.sup.6    Preparation No. 70    Whole     4000                  17.4                      2.9 × 10.sup.3                           100 1.7 × 10.sup.2                                    1    culture.sup.a    10        0.5 0.010                      0.6 × 10.sup.6                           3   0.6 × 10.sup.8                                    0.4 × 10.sup.6    11        0.5 0.008                      2.5 × 10.sup.6                           11  3.2 × 10.sup.6                                    1.9 × 10.sup.6    Preparation No. 71/72    Whole     8300                  14.5                      1.0 × 10.sup.4                           100 7.1 × 10.sup.2                                    1    culture.sup.a    10        0.5 0.035                      0.2 × 10.sup.6                           0.1 0.1 × 10.sup.8                                    0.7 × 10.sup.4    11        0.5 0.026                      2.0 × 10.sup.7                           11  7.6 × 10.sup.6                                    1.1 × 10.sup.6    12        0.5 0.020                      1.8 × 10.sup.7                           11  9.2 × 10.sup.6                                    1.3 × 10.sup.6    13        0.5 0.014                      0.4 × 10.sup.7                           2   2.5 × 10.sup.6                                    0.4 × 10.sup.6    __________________________________________________________________________     .sup.a Assays of protein (280 nm absorbance) and of mitogenicity     (lymphocyte proliferation) of starting material were actually performed o     ultrafiltered culture supernatants. As for the remainder of the scheme,     cellular debris was removed with the first step of 50%saturated ammonium     sulfate precipitation.

FIG. 3 shows homogeneity of MAM in the ultimate chromatographic fractionaccording to SDS-polyacrylamide gradient gel electrophoresis.Electrophoresis, staining, and other procedures were formed onPharmacia-LKB PAA 4/30 precast gels according to P. Lambin et al., 74Anal. Biochem. 567 (1976). Left to right, the lanes contained: 20 μghorse heart cytochrome c (Sigma Chemical, St. Louis, Mo.); 1 μg each ofBio-Rad (Hercules, Calif.) standard proteins; 5 μg bovine erythrocytecarbonic anhydrase (Sigma); 60 μl of final hydrophobic interactionchromatographic fraction 14, preparation 69, containing about 1 μg MAM;and 3 μg each of Pharmacia-LKB standard proteins. Homogeneity wassimilarly demonstrated (but not shown here) in the familiar non-gradientSDS-polyacrylamide gel electrophoretic system of U. Laemmli, 227 Nature680 (1970). Homogeneity of this fraction was further demonstrated (butnot shown here) by high and unique residue yield at each early step ofamino acid sequencing by Edman degradation, described below.

The biological activity of homogeneous MAM is heat labile and proteaselabile. By comparison to globular molecular weight standard proteins,homogeneous MAM gave a molecular weight estimate of 27,000 Daltons ineach of two gel filtration methods (on Sephadex G-50 or Superose 12,both from Pharmacia-LKB) and in two denaturing gel electrophoreticmethods (described above). The molecular weight of MAM had beenpreviously been reported to be about 15,000 as estimated by gelfiltration. C. Atkin et al., 137 J. Immunol. 1581 (1986). The earliermethod resulted in only partial (≦200-fold) purification of MAM, whereasthe method reported herein results in vastly greater purification (seenext paragraph) to apparent homogeneity. The reported molecular weightof MAM of 15,000 may have represented degraded but still active MAMmolecules, but more likely was an artifact of delayed elution from thegel filtration resin because of MAM-resin adsorption under conditions ofmuch lower ionic strength than used here. The apparent isoelectric pointof MAM is estimated as pI>9.0 from isoelectric focusing.

The essentially homogeneous MAM retains all the biological activitiesnoted above for crude M. arthritidis culture supernatants, includingV.sub.β -specific proliferation of T cells, induction of interleukin-2production,and in vivo immunosuppressive properties. Half-maximalmitogenic activity for genetically appropriate T-cells occurs at about10⁻¹⁴ M MAM. The specific mitogenic activity of homogeneous MAM formurine lymphocytes according to MLPA (about 1×10⁸ U/mg) corresponds toabout a million-fold purification of the mitogenic activity of crudeculture supernatants (about 100 U/mg culture supernatant proteins).

The combination of using a high-MAM-yielding strain of M. arthritidis,propagating the organisms in "boiled medium," back extraction of thematerial that was insoluble in 80% saturated (NH₄)₂ SO₄, and especiallygel filtration at maximum ionic strength short of salting out MAM,yielded fractions that could be efficiently purified by cationicexchange chromatography. No other combination of steps has been found toyield homogeneous preparations of MAM. It is thought that these stepspermit purification of homogeneous MAM because they minimize formationof intractable complexes of MAM with nucleic acids and other highmolecular weight components of the starting culture.

Amino Acid Sequence of Purified MAM

The amino acid sequence of the N-terminal region of MAM, purifiedaccording to the procedure described above, was determined by a pulsedliquid phase method with a BioBreen-treated fiber glass filter samplesupport based on the Edman degradation method. P. Edman & G. Begg, 1Eur. J. Bioch. 80 (1967); T. Hugli, Techniques in Protein Chemistry(1989); P. Matsudaira, A Practical Guide to Protein and PeptidePurification for Microsequencing (1989). The machine used was an AppliedBiosystems, Inc. (ABI, Foster City, Calif.) Model 477A ProteinSequencer. Using this procedure the sequence of the N-terminal region ofmature MAM protein was obtained, which is identified as SEQ ID NO:1.

Nucleotide Sequence of MAM Gene

A genomic library of M. arthritidis PG-6 was prepared according tostandard methods in the EMBL3 phage vector. M. arthritidis genomic DNAwas partially digested with the restriction endonuclease SalI and thenligated into SalI-digested EMBL3 vector DNA. The resulting library ofclones was screened for the MAM gene by Southern hybridization (or"Southern blotting," E. Southern, 98 J. Mol. Biol. 503(1975); for modernelaboration of this technique see J. Sambrook et al., Molecular Cloning§§ 9.31-9.59 (2d ed., 1989)) with the 5'-³² P-phosphorylated syntheticdegenerate oligodeoxynucleotide

    ______________________________________    GAAAAYCCAA AAAAAGCWCA AAAACA                           26    (SEQ ID NO:2)    ______________________________________

that was designed by reverse translation (according to the codon usagetable of Muto, 23 Israel J. Med. Sci. 334 (1987) for Mycoplasmacapricolium) to match as closely as possible the actual coding sequencefor amino acids 6-14 of SEQ ID NO:1. This and otheroligodeoxynucleotides were synthesized using an ABI Model 380B DNASynthesizer with proprietary phosphoramidite chemistry.

Four positive recombinant clones were selected for furthercharacterization, clones 1246, 1410, 1722, and 1903. Southern analysisof these four indicated that a 4 kb HindIII fragment, a 3 kb BamHIfragment, and a 1.6 kb Sau3AI fragment probably contained the entire MAMgene. These restriction fragments were subcloned into pBSSK³⁰ and pTZ18Rvectors as follows. Clone pBSMAM-1410-Sau3AI is a subclone of the 1.6 kbSau3AI fragment of clone 1410 in pBSSK11⁺. Clones pTZMAM-1722-He andpTZMAM-1722-Hn are subclones, in opposite orientations, of the 4 kbHindIII fragment of clone 1722 in pTZ18R. Nucleotide sequencing of thesesubclones with the Sanger dideoxynucleotide chain termination method, F.Sanger et al., 74 Proc. Nat'l Acad. Sci. USA 5463 (1977), yielded asequence of the entire MAM gene, SEQ ID NO:3.

The sequenced MAM gene contains a region that, when translated, gives anamino acid sequence identical to that determined by direct amino acidsequencing (amino acids 1-54). This N-terminal region of the mature MAMprotein is preceded by a 39 amino acid residue signal peptide orpre-peptide. Mature MAM (SEQ ID NO:4) contains 213 amino acid residues,which is calculated to have a molecular weight of 25,294 and a pI of10.1.

Identification of a Peptide with Biological Activity

To confirm that the amino acid sequence determined by N-terminalsequencing of the homogeneous protein preparation was the sequence of aprotein having MAM activity, three peptides were synthesized and testedfor ability to block the mitogenic activity of intact MAM protein bycompetitive inhibition. The peptides chosen were SEQ ID NO:5, SEQ IDNO:6, and SEQ ID NO:7 which include, respectively, amino acids 1-13,27-44, and 15-32 of mature MAM. These and other peptides weresynthesized on an ABI Model 431A Peptide Synthesizer using proprietaryFmoc-based modified Merrifield technique. In trials, each of thepeptides was pre-incubated with genetically responsive murinesplenocytes for 1 hour prior to the addition of MAM. Lymphocyteactivation was measured by a standard procedure using incorporation oftritiated thymidine (³ H-TdR) into replicated DNA. B. Cole, 2 Methods inMycoplasmology 389 (1983). Similar results were obtained from mitogen attwo concentrations: homogeneous MAM preparation 71/72, fraction 12, at1:25,000- and 1:250,000-fold dilutions corresponded to approximate MAMconcentrations of 0.8 ng/ml and 0.8 pg/ml, or 30 pM and 30 fM,respectively. As shown in FIG. 4, SEQ ID NO:7 was inhibitory forMAM-induced proliferation whereas SEQ ID NO:5 and SEQ ID NO:6 were not.This result strongly suggests that the N-terminal sequence is indeedthat of MAM and not of a contaminant. Further, amino acids 15-32 appearto comprise a domain of mature MAM that has a biological activity. Noneof the peptides had any effect on concanavalin A-induced lymphocytestimulation.

These results further suggest that if MAM or MAM-like amino acidsequences present in other superantigens are responsible for triggeringor causing the flares in commonly seen human autoimmune diseases, thentherapeutic interventions may be developed. One such therapeuticintervention would comprise administering synthesized peptidescorresponding to these active domains of the superantigen tocompetitively block lymphocyte activation by the intact superantigen.Another therapeutic intervention would comprise immunizing individualsagainst intact superantigens or against those superantigen domains thatare active in initiation of the disease process. Immunization ortreatment of patients with peptides, selected for their regions ofsequence similarity between MAM and other superantigens--even if notdirectly involved in lymphocyte activation, may induce an immuneresponse to the superantigen that blocks all of its biologicalactivities.

Sequence Comparisons of MAM to other Superantigens

The amino acid sequence of MAM, as deduced by direct amino acidsequencing of the N-terminal region of MAM (SEQ ID NO:1) together withdeducing the entire amino acid sequence of MAM from the nucleotidesequence of the MAM gene (SEQ ID NO:4), was compared to amino acidsequences of other superantigens. The purpose of these comparisons wasto identify oligopeptide segments that are homologous between MAM andthese other superantigens. The rationale is that the presence ofhomologous oligopeptide segments may reflect similar functionalities orbinding sites within this structurally diverse group of proteins. Thenature of the analysis does not require overall sequence similarity. Anysequential ordering of similar oligopeptide segments may be detected inthe MAM-superantigen being compared.

Sequence comparisons were made by computer using the DDMATRIX programobtained from Intelligenetics (Mountain View, Calif.). MAM tosuperantigen comparisons were made by comparison of all possible 23-meror 24-mer combinations. Rather than search for exact identities of aminoacid sequence segments, conservative substitutions of amino acidresidues were permitted according to the scheme of M. Jimenez-Montano &L. Zamora-Cortina, Evolutionary model for the generation of amino acidsequences and its application to the study of mammal alpha-hemoglobinchains, Proc. VIIth Int'l Biophysics Congress, Mexico City (1981).Conservative substitution groups are shown in the following table.

                  TABLE 2    ______________________________________    Residue or Group                 Physical Character    ______________________________________    Pro          Proline    Ala, Gly     Minimal side chains, either hydrophobic or                 hydrophilic    Ser, Thr     R-OH side chains, hydrophilic    Asn, Gln     Amide side chains, hydrophilic    Asp, Glu     Carboxylic acid side chains, hydrophilic    His          Histidine, basic    Lys, Arg     Alkyl amine side chains, basic    Cys          Cysteine    Ile, Leu, Met, Val                 Hydrophobic alkyl side chains    Phe, Trp, Tyr                 Aromatic    ______________________________________

The amino acid sequence of MAM was compared to the following bacterialsuperantigen amino acid sequences: toxic shock syndrome toxin 1 (TSST-1,GenBank accession number J02615, D. A. Blomster-Hautamaa et al., 261 J.Biol. Chem. 15783 (1986)); staphylococcal enterotoxin A (SEA, GenBankaccession number M18970, M. J. Betley and J. J. Mekalonos, 170 J.Bacteriol. 34 (1988)); staphylococcal enterotoxin B (SEB, GenBankaccession number M11118, C. L. Jones and S. A. Khan, 166 J. Bacteriol.29 (1986)); staphylococcal enterotoxin C1 (SEC1, GenBank accessionnumber X05815, G. A. Bohach and P. M. Schlievert, 209 Mol. Gen. Genet.15 (1987)); streptococcal pyrogenic exotoxin A (SPEA, GenBank accessionnumbers SP156SPEA etc. for allele 1, SP250SPEA etc. for allele 2,SP158SPEA etc. for allele 3, and SP262SPEA for allele 4, K. Nelson etal., 174 J. Exp. Med. 1271 (1991)); and streptococcal pyrogenic exotoxinC (SPEC, GenBank accession number M35514, S. C. Goshorn and P. M.Schlievert, 56 Infect. Immun. 2518 (1988)).

The amino acid sequence of MAM was also compared to the amino acidsequences of retroviral superantigens, including: murine tumor minorlymphocyte-stimulating (Mls) antigens MTV7 (GenBank accession no.M90535, U. Beutner et al., 89 Proc. Natl. Acad Sci. USA 5432 (1992)),MTV1, MTV9, MTV8, MTV11, MMTV C3H, MMTV BR6, and MMTV GR (Beutner,supra); HIV-1 envelope polyprotein (GenBank accession no. M15896, A.Srinivasan et al., 5 AIDS Res. Hum. Retroviruses 121-129 (1989)); andHIV-1 GAG protein (GenBank accession no. M17451, B. R. Starcich et al.,45 Cell 637-648 (1986)). The results of these comparisons are summarizedin FIGS. 5-7. In FIG. 5, solid lines indicate small regions of the aminoacid sequence of MAM (SEQ ID NO:4) that resemble regions ofGram-positive bacterial and retroviral superantigens. Thesesuperantigens were considered to share a potential epitope with MAMwhen, allowing for conservative substitutions of amino acid residues(according to the scheme of Jimenez-Montano & Zamora-Cortina, supra),each protein being compared had at least a common tetrapeptide sequencewith additional common residues separated by no more than threedissimilar amino acids. In FIG. 6, sequence similarities of Grampositive bacterial superantigens to potential epitopic "Region 1" of MAMare shown. The boxed portion of SEQ ID NO:8 corresponds to theoligopeptide able to block lymphocyte proliferation, SEQ ID NO:7. Otherboxed amino acids in SEQ ID NO:9 through SEQ ID NO:14 show regions ofconservative amino acid similarity. These results show that there arenumerous examples of conservative amino acid sequence similaritiesbetween MAM and other superantigens. Some of these similarities may nothave biological significance. However, it is noteworthy that withallowance of conservative residue substitutions and minimal gaps andinsertions, the 17-residue motif of MAM(16-32) and of SEQ ID NO:15, ValGln Asn Leu Asn Asn Val Val Phe Thr Asn Lys Glu Leu Glu Asp Ile, issegmentally but strongly represented in both bacterial and (see nextsection) murine retroviral antigens. It is further noteworthy that thisSEQ ID NO:15, embodied as all but the first and last residues ofsynthetic peptide SEQ ID NO:7, was shown to be capable of inhibitingMAM-induced cell proliferation (FIG. 4). This result suggests thatsequence comparisons may be beneficial in selecting candidate peptidesfor blocking or immunizing against superantigens responsible for humanautoimmune disease.

Sequence Comparisons of MAM to Human Immunodeficiency Virus (HIV)

Retroviruses have been postulated to play a role in human rheumaticdiseases. Further, antibodies to human T-cell leukemia virus 1 (HTLV-1)and to human immunodeficiency virus 1 (HIV-1) have been detected in serafrom patients with lupus and autoimmune diseases. Since lupus patientsdid not harbor HIV nucleic acid, it is likely that the antibodies thatwere detected were directed to an agent that bears antigenic componentssimilar to some components of HIV.

Superantigens may play a role in AIDS. HIV, the causal agent of AIDS,can only replicate in activated lymphocytes. Superantigens activate Tlymphocytes, thereby providing the virus more cells in which it canmultiply. Evidence for the existence of a superantigen in HIV is thatHIV replicates better in T cells expressing the V.sub.β 12 and V.sub.β17 human T cell receptors than it does in other T cell subsets. J.Laurence et al., 358 Nature 255 (1992). Importantly, human V.sub.β 12and V.sub.β 17 are among the subsets that are activated by MAM, and areequivalent to the murine V.sub.β 8 and V.sub.β 6 T cell receptors.

Sequence comparisons of MAM with known sequences of HIV proteins revealthe existence of a number of similar regions, FIG. 7. In FIG. 7,sequence similarities of retroviral superantigens and HIV-1 proteins topotential epitopic "Region 1" of MAM are shown. The boxed portion of SEQID NO:16 corresponds to the oligopeptide able to block lymphocyteproliferation, SEQ ID NO:7. Other boxed amino acids in SEQ ID NO:17through SEQ ID NO:23 show regions of conservative amino acid similarity.Some of these regions of sequence similarity are also similar tosequence domains in the murine retroviral superantigens. Thus the motifsVal Gln Asn Leu Gln Gly Gln Met (SEQ ID NO:24) (HIV-GAG) and Val Gln GlnGln Asn Asn Leu Leu (SEQ ID NO:25) (HIV Z321, envelope) are similar toboth mouse MTV-7 retroviral superantigen (Val Gln Asp Tyr Asn Leu AsnAsn; SEQ ID NO:26) and to MAM (SEQ ID NO:15) (FIG. 7). The latter motifis duplicated in the biologically active peptide (SEQ ID NO:7) that wassynthesized to correspond to amino acid residues 15 to 32 of MAM.Comparison of MAM sequences to those of HIV may therefore detect theexistence of HIV superantigens.

Thus, if regions of sequence similarity between MAM and HIV, HTLV-1, orother viruses are regions important in inducing immunodysfunction, thenprotective strategies may be used against the potential harmful effectsof these superantigens. Such protective strategies include immunizingthe host with peptides that include these sequences or administeringpeptides that bear these sequences.

Sequence Comparisons of MAM to Other Proteins

Sequence similarities have also been detected between MAM and a numberof other proteins including some regulatory proteins. MAM or MAMpeptides may also be used to enhance or block the action of theseregulatory proteins.

Utility of the Invention

Homogeneous preparations of MAM or specific MAM peptides may be used asreagents to modify immune function in vittro and in vivo as exemplifiedby the following examples.

EXAMPLE 1

MAM may be used for detecting or selecting (isolating) specific V.sub.β-chain TCR-bearing T cells in vitro or in vivo. B. Cole et al., Clin.Infect. Dis. (in press, 1993); B. Cole et al., 150 J. Immunol. 3291(1993); L. Baccala et al., 35 Arthritis Rheum. 434 (1992).

Lymph node cells were suspended at 1.25×10⁶ /ml in 2 ml 24-well platesin RPMI 1640 medium supplemented with 200 mM L-glutamine, 5% humanserum, 2.5×10⁻⁵ M 2-mercaptoethanol, 0.11% sodium pyruvate, 0.1 mMnonessential amino acids (GIBCO Laboratories, Chicago Falls, Ohio), 50U/ml penicillin G, and 50 μg/ml streptomycin sulfate. Lymphocytecultures were incubated with MAM (10⁷ to 5×10⁷ U/ml) at 1:5000 for 3 to5 days. As controls, lymphocytes were unstimulated or incubated for 3days with 5 μg/ml concanavalin A. The cultures were then harvested, andthe dead cells were removed by Ficoll gradient centrifugation.

A set of 17 riboprobes for analysis of V.sub.β^(b) haplotype mice wasprepared as previously described, P. Singer et al., 170 J. Exp. Med.1869 (1989), which is hereby incorporated by reference. A new set of 8V.sub.β riboprobes for analysis of V.sub.β^(a) haplotype mice (strainsC57BR and SWR) was also made. This V.sub.β^(a) haplotype-specific probeset lacks riboprobes for the 9 V.sub.β (5.1, 5.2, 8.1, 8.2, 8.3, 9, 11,12, 13) that are genetically deleted in V.sub.β^(a) mice, and includesnew V.sub.β^(a) probes for V.sub.β 1, 3, and 6, which are polymorphicbetween V.sub.β^(b) and V.sub.β^(a) haplotype mice. The principles ofthe multiprobe RNase protection assay have been detailed in P. Singer etal., 170 J. Exp. Med. 1869 (1989). In brief, RNA (5 to 10 μl) extractedfrom cell pellets and lyophilized was dissolved in hybridization buffer(5 μl of 80% formamide, 0.4M NaCl, 1 mM EDTA, and 40 mM PIPES, pH 6.7)containing the appropriate riboprobe set (1×10³ cpm×number of uridineresidues in the riboprobe set). The solution was overlaid in mineral oiland incubated at 56° C. for 12 to 16 hours. Unhybridized probe andtarget RNA was digested with RNase A (5 μg/ml) and RNase T1 (10 U/ml) in50 μl digestion buffer (10 mM Tris, pH 7.5, 5 mM EDTA, and 0.3M NaCl).After 1 hour at 30° C., digested samples containing the "protected"probe-target duplexes were phenol-chloroform extracted, ethanolprecipitated, dissolved in sample buffer, and electrophoresed instandard 6% polyacrylamide-urea sequencing gels. Autoradiography of thedried gel was done on Kodak XRP film at -70° C. with intensifying screenfor 10 to 20 hours. An Ambis radioanalytic imaging apparatus (AmbisSystems, San Diego, Calif.) was used to quantitate V.sub.β transcriptlevels. The net cpm at a given band corresponding to a specificprotected V.sub.β probe was calculated by the formula ((cpm of V.sub.β-specific band)--(cpm background around the band)--(number of uridineresidues on the specific V.sub.β probe)); this value was then expressedas the percentage of the V.sub.β included in the probe set.

Lymph node cells from two V.sub.β^(b) haplotype strains,CBA/CaJ.(H-2^(k), Mls^(b)) and CBA/J (H-2^(k), Mls^(a+c)) were grown inthe presence of MAM for 3 to 5 days and viable activated cells collectedover a Ficoll gradient. The results with CBA/CaJ lymphocytes (Table 3)show that MAM activates T cells bearing V.sub.β 6, 8.1, 8.2, and 8.3.Slightly different results were obtained with cells from CBA/J mice,which show somatically imposed clonal deletions/depletions of cellsbearing V.sub.β 3.1, 5, 6, 7, 8.1, 9, 11, 12, and 16 because of thepresence of various MTV integrants on several chromosomes.

                  TABLE 3    ______________________________________            % V.sub.β Transcripts in            CBA/CaJ Cells    CBA/J Cells    V.sub.β Tested              Control MAM        Control                                       MAM    ______________________________________    1         2.2     0.4        2.3   0.6    2         9.5     1.8        10.8  0.8    3.1       3.7     2.1        0.1   0.2    4         1.8     0.4        3.1   0.3    5.1       3.9     3.6.sup.a  2.2   3.6    5.2       0.2     0.2        0.2   0.7    6         2.8     5.6        0.3   0.2    7         0.8     0.4        1.0   0.4    8.1       20.2    30.8       8.0   17.8    8.2       17.1    34.0       18.8  37.8    8.3       16.5    15.6       24.0  31.6    10        2.6     0.5        2.1   0.4    11        0.8     0.4        2.2   1.1    12        1.2     1.0        0.7   1.0    13        4.3     1.1        7.3   0.9    14        8.7     1.5        12.1  1.4    15        4.1     0.4        4.7   0.5    ______________________________________     .sup.a V.sub.B used by MAM are underlined.

EXAMPLE 2

MAM may be used for modifying immune responses in vivo by suppressing orchanging specific T or B lymphocyte functions. B. Cole & D. Wells, 58Infect. Immun. 228 (1990); B. Cole et al., Clin. Infect. Dis. (in press,1993).

Table 4 shows that MAM promotes survival of transplants, exemplifyingMAM's ability to suppress T cell functions. Detailed methodology forskin grafting has been described in D. Steinmuller, 108 Meth. Enzymol.20 (1984), which is hereby incorporated by reference. BALB/c skin graftsabout 1 cm in diameter were taken from the ear and cleaned of cartilageand fat. Grafts were made onto 1 cm beds of vascular fascia of theflanks of C3H/HeJ mice. C3H mice received only two or three injectionsof MAM diluted 1:20 or PBS before receiving a graft from a BALB/c mouse.Mice that received two (experiment 2) or three (experiment 3) injectionsof MAM showed a significant prolongation of graft survival as comparedwith the survival of mice that received PBS.

                  TABLE 4    ______________________________________    Material injected                  Survival of BALB/c    into C3H recipient                  skin (days).sup.a                               Significance    ______________________________________    Experiment 1    PBS × 1 8.6 ± 1.3 NS.sup.b    MAM × 1 10.0 ± 1.6    Experiment 2    PBS × 2 9.7 ± 1.8 P < 0.05    MAM × 2  12 ± 1.5    Experiment 3    PBS × 3 10.3 ± 0.6                               P < 0.05    MAM × 3 14.6 ± 2.7    ______________________________________     .sup.a Values are the means for six mice per group ± SD.     .sup.b Not significant.

Table 5 shows the effects of MAM on humoral antibody responses toovalbumin (OVA) as measured in terms of lymphocyte proliferation,exemplifying enhancement of B cell functions of MAM in vivo. The methodused for stimulation of lymphocytes is detailed in B. Cole et al., 144J. Immunol. 425 (1990), which is hereby incorporated by reference.Proliferative responses of lymphocytes to OVA were measured by amodification of the referenced lymphocyte proliferation assay. CBA orC3H mice were injected in the rear footpads with 50 μg of OVA emulsifiedwith complete Freund's adjuvant. After 12 days, draining lymph nodeswere harvested and lymphocytes were tested for proliferative responsesto OVA with use of RPMI 1640 medium supplemented with 2 mM L-glutamine,5% heat-inactivated human serum, 5×10⁻⁵ M 2-mercaptoethanol, 0.011%sodium pyruvate, 1% nonessential amino acids (×100), penicillin, andstreptomycin.

                  TABLE 5    ______________________________________                 Specific uptake of  .sup.3 H!thymidine (cpm ×                 10.sup.3).sup.a in response to OVA at indicated                 concentration (μg/ml).sup.b    Injection schedule                   Experiment 1 Experiment 2    Day 0    Day 2     50       250   50     250    ______________________________________    PBS      OVA       5.8      9.0   1.5   2.1    MAM 1:10 OVA       34.0     29.1  8.3   14.9    PBS +    --        9.8      18.3  1.0   3.4    OVA    MAM 1:10 +             --        27.6     37.7  5.9   16.8    OVA    OVA      PBS       3.0      3.3   7.0   10.2    OVA      MAM 1:10  23.6     39.5  27.5  34.7    ______________________________________     .sup.a Uptake of  .sup.3 H!thymidine in response to OVA minus uptake in     absence of OVA.     .sup.b Values are the means for two separate mice tested in triplicate.

EXAMPLE 3

MAM may be used for triggering, enhancing, exacerbating, or otherwisealtering the course of autoimmune arthritis thereby providing anexperimental model system for the study of the etiology and mechanismsof development of autoimmune diseases. B. Cole & M. Griffiths, 36Arthritis Rheum. 994 (1993).

Table 6 shows the effects of MAM, SEA, and SEB on the development ofCIA. SEB was chosen since, as with MAM, it activates T cells bearing theV.sub.β 8 chain segments of the TCR. SEA was also used since it fails toactivate any of the T cell subsets that have been shown or thought tomediate CIA (i.e., T cells bearing V.sub.β 5, 6, 7, 8, and 9). In thisexperiment, B10.RIII mice, a very susceptible strain (100% incidencewith injection of ≦100 μg native porcine type II collagen (PII)), weresuboptimally immunized with 5 μg PII. PII was prepared as previouslydescribed in M. Griffiths et al., 24 Arthritis Rheum. 781 (1981), whichis hereby incorporated by reference. PII was dissolved overnight in 0.1Nacetic acid and was then emulsified with 50% Freund's complete adjuvant.Two to four-month-old mice were immunized with 5 μg of PII emulsionintradermally in the base of the tail. The mice were challenged 16 dayslater with intravenous injections of 0.2 ml PBS, 1:35 MAM, or SEB or SEA(5 μg/mouse). It had previously been established that SEB and SEA indoses of 5 μg/mouse induce lymphadenopathy and splenomegaly as early as1-2 days post-injection.

                  TABLE 6    ______________________________________                                    Day of maximum               Arthritis Day of onset,                                    severity, mean ±    IV treatment               incidence mean ± SD                                    SD    ______________________________________    PBS, 0.2 ml               1/10      5.0        31.0    MAM 1:35, 0.2 ml               5/10      8.0 ± 7.9                                    15.6 ± 7.8    SEB, 5 μg               5/10       8.0 ± 10.2                                    14.8 ± 9.0    SEA, 5 μg               2/10      14.0 ± 5.7                                    28.5 ± 3.5    ______________________________________

Only one of the control animals receiving PBS developed arthritis, andthe severity gradually increased until termination of the experiment at31 days post-challenge. Mice receiving either MAM or SEB exhibited a 50%incidence of arthritis, with an early onset time. In fact, among micetreated with MAM, 3 of 10 showed arthritis just 3 days post-injection.Mean maximum arthritis severity for MAM-injected animals occurred at day15. In contrast, SEA induced a late onset arthritis occurring in only20% of the mice and, as in PBS-injected animals, the arthritis severitydid not peak until close to termination of the experiment. Thus, thesedata provide evidence that MAM triggers an early onset of CIA.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 26    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 54 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: no    (iv) ANTI-SENSE: no    (v) FRAGMENT TYPE: N-terminal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Mycoplasma arthritidis    (B) STRAIN: PG6    (G) CELL TYPE: unicellular organism    (ix) FEATURE:    (A) NAME/KEY: N-terminal region of mature protein    (B) LOCATION: 1 to 54    (C) IDENTIFICATION METHOD: direct sequencing of    protein    (D) OTHER INFORMATION: contains domain that blocks    cell proliferation    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    MetLysLeuArgValGluAsnProLysLysAlaGlnLysHisPheVal    151015    GlnAsnLeuAsnAsnValValPheThrAsnLysGluLeuGluAspIle    202530    TyrAsnLeuSerAsnLysGluGluThrLysGluValLeuLysLeuPhe    354045    LysLeuLysValXaaGln    50    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (iii) HYPOTHETICAL: yes    (iv) ANTI-SENSE: no    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Mycoplasma arthritidis    (B) STRAIN: PG6    (G) CELL TYPE: unicellular organism    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    GAAAAYCCAAAAAAAGCWCAAAAACA26    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1107    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: genomic DNA    (iii) HYPOTHETICAL: no    (iv) ANTI-SENSE: no    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Mycoplasma arthritidis    (B) STRAIN: PG6    (G) CELL TYPE: unicellular organism    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    TTTAACACTTCTTTCGGTTATTAATAACTTTAAATTCTAATTAAATTGGTAAAGCGGGTA60    AACAAAGAAACTATTTAAAAATTTATGAAATTAATATTTAACTTTATAAAATAAAATTTC120    GCTGTGAAAATGAAATTCTTCACAAATTTAAAAATCATAAGGAATAAAAAA171    MetLysPhePheThrAsnLeuLysIleIleArgAsnLysLys    35-30    ATGAAAACAAAAAAATTATTAATCGCAACCGTCACTTTAGCGACTGGG219    MetLysThrLysLysLeuLeuIleAlaThrValThrLeuAlaThrGly    25-20-15-10    CTTTTAGGAATTTTACCATTAACTAGCATGAAACTTAGAGTTGAAAAT267    LeuLeuGlyIleLeuProLeuThrSerMetLysLeuArgValGluAsn    515    CCTAAAAAAGCTCAAAAGCATTTTGTGCAAAATTTAAATAATGTTGTA315    ProLysLysAlaGlnLysHisPheValGlnAsnLeuAsnAsnValVal    101520    TTTACTAATAAAGAGCTTGAAGATATCTACAATTTAAGTAATAAAGAA363    PheThrAsnLysGluLeuGluAspIleTyrAsnLeuSerAsnLysGlu    253035    GAAACAAAAGAAGTATTAAAATTGTTTAAATTGAAGGTCAACCAATTT411    GluThrLysGluValLeuLysLeuPheLysLeuLysValAsnGlnPhe    40455055    TATAGACATGCTTTTGGTATAGTGAATGACTACAATGGACTTCTTGAA459    TyrArgHisAlaPheGlyIleValAsnAspTyrAsnGlyLeuLeuGlu    606570    TACAAAGAAATTTTTAATATGATGTTTTTAAAATTAAGCGTTGTCTTT507    TyrLysGluIlePheAsnMetMetPheLeuLysLeuSerValValPhe    758085    GACACACAACGTAAAGAGGCAAATAACGTCGAACAAATCAAAAGAAAT555    AspThrGlnArgLysGluAlaAsnAsnValGluGlnIleLysArgAsn    9095100    ATCGCTATTTTAGATGAAATAATGGCAAAAGCAGATAACGATTTATCT603    IleAlaIleLeuAspGluIleMetAlaLysAlaAspAsnAspLeuSer    105110115    TACTTTATATCTCAGAATAAGAATTTTCAAGAGTTATGAGATAAAGCT651    TyrPheIleSerGlnAsnLysAsnPheGlnGluLeuTrpAspLysAla    120125130135    GTCAAACTAACTAAAGAAATGAAAATAAAACTTAAATTCCAAAAACTA699    ValLysLeuThrLysGluMetLysIleLysLeuLysGlyGlnLysLeu    140145150    GATCTTCGTGATGGTGAAGTTGCAATAAACAAAGTAAGAGAATTATTT747    AspLeuArgAspGlyGluValAlaIleAsnLysValArgGluLeuPhe    155160165    GGCAGCGACAAAAATGTAAAAGAGCTTTGATGATTTAGATCTCTTCTA795    GlySerAspLysAsnValLysGluLeuTrpTrpPheArgSerLeuLeu    170175180    GTAAAAGGTGTTTACCTTATAAAACGCTATTACGAAGGTGATATTGAA843    ValLysGlyValTyrLeuIleLysArgTyrTyrGluGlyAspIleGlu    185190195    CTTAAAACGACATCGGATTTTGCAAAAGCTGTTTTTGAAGAT885    LeuLysThrThrSerAspPheAlaLysAlaValPheGluAsp    200205210    TAATATTAAACATATATAACAAATTATCCCCCCAATCTAAAAGGTTGGGGGGATTTTAAA945    TAAATTCCTTGCATCTAGCAAGGATAAATAAGATAGAAATAAATTGGTTAGTTAAAAAAT1005    GTTTGGTCCGTTGCAATTATGATTTTTTCGTTTTGTATTGTAATTGGCACTTCGCTATAT1065    TCCTTTATTTTTCCAGAAATAATTTCCATAGCAAGCATGTTT1107    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 213 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Mycoplasma arthritidis    (B) STRAIN: PG6    (G) CELL TYPE: unicellular organism    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    MetLysLeuArgValGluAsnProLysLysAlaGlnLysHisPheVal    151015    GlnAsnLeuAsnAsnValValPheThrAsnLysGluLeuGluAspIle    202530    TyrAsnLeuSerAsnLysGluGluThrLysGluValLeuLysLeuPhe    354045    LysLeuLysValAsnGlnPheTyrArgHisAlaPheGlyIleValAsn    505560    AspTyrAsnGlyLeuLeuGluTyrLysGluIlePheAsnMetMetPhe    65707580    LeuLysLeuSerValValPheAspThrGlnArgLysGluAlaAsnAsn    859095    ValGluGlnIleLysArgAsnIleAlaIleLeuAspGluIleMetAla    100105110    LysAlaAspAsnAspLeuSerTyrPheIleSerGlnAsnLysAsnPhe    115120125    GlnGluLeuTrpAspLysAlaValLysLeuThrLysGluMetLysIle    130135140    LysLeuLysGlyGlnLysLeuAspLeuArgAspGlyGluValAlaIle    145150155160    AsnLysValArgGluLeuPheGlySerAspLysAsnValLysGluLeu    165170175    TrpTrpPheArgSerLeuLeuValLysGlyValTyrLeuIleLysArg    180185190    TyrTyrGluGlyAspIleGluLeuLysThrThrSerAspPheAlaLys    195200205    AlaValPheGluAsp    210    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: N-terminal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Mycoplasma arthritidis    (B) STRAIN: PG6    (G) CELL TYPE: unicellular organism    (ix) FEATURE:    (A) NAME/KEY: N-terminus of mature MAM, with a Cys    residue added at the N-end    (B) LOCATION: Cys at N-terminal followed by MAM    residues 1- 13    (D) OTHER INFORMATION: does not inhibit cell    proliferation    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    CysMetLysLeuArgValGluAsnProLysLysAlaGlnLys    1510    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment plus C-terminal Cys    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Mycoplasma arthritidis    (B) STRAIN: PG6    (G) CELL TYPE: unicellular organism    (ix) FEATURE:    (B) LOCATION: MAM residues 27-44, plus C-terminal Cys    (D) OTHER INFORMATION: does not inhibit cell    proliferation    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    LysGluLeuGluAspIleTyrAsnLeuSerAsnLysGluGluThrLys    151015    GluValCys    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment plus C-terminal Cys    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Mycoplasma arthritidis    (B) STRAIN: PG6    (G) CELL TYPE: unicellular organism    (ix) FEATURE:    (A) NAME/KEY: competitively inhibits MAM-induced cell    proliferation    (B) LOCATION: MAM residues 15 to 32, plus C-terminal    Cys    (C) IDENTIFICATION METHOD: in vitro competitive    inhibition assay    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    PheValGlnAsnLeuAsnAsnValValPheThrAsnLysGluLeuGlu    151015    AspIleCys    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Mycoplasma arthritidis    (B) STRAIN: PG6    (G) CELL TYPE: unicellular organism    (ix) FEATURE:    (B) LOCATION: MAM residues 12 to 38    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    GlnLysHisPheValGlnAsnLeuAsnAsnValValPheThrAsnLys    151015    GluLeuGluAspIleTyrAsnLeuSerAsnLys    2025    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Staphylococcus aureus    (B) STRAIN: S6    (G) CELL TYPE: unicellular organism    (ix) FEATURE:    (A) NAME/KEY: staphylococcal enterotoxin B amino acid    sequence having sequence similarity to    MAM.    (B) LOCATION: residues 39 to 65    (C) IDENTIFICATION METHOD: computer searching for    sequence similarities.    (x) PUBLICATION INFORMATION:    (A) AUTHORS: Jones, C.L.    Khan, S.A.    (B) TITLE: Nucleotide Sequence of the Enterotoxin B    Gene from Staphylococcus aureus    (C) JOURNAL: J. Bacteriol.    (D) VOLUME: 166    (E) ISSUE: 1    (F) PAGES: 29-33    (G) DATE: APR-1986    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    LysSerIleAspGlnPheLeuTyrPheAspLeuIleTyrSerIleLys    151015    AspThrLysLeuGlyAsnTyrAspAsnValArg    2025    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Staphylococcus aureus    (B) STRAIN: S6    (G) CELL TYPE: unicellular organism    (ix) FEATURE:    (A) NAME/KEY: staphylococcal enterotoxin B amino acid    sequence having sequence similarity to    MAM.    (B) LOCATION: residues 56 to 82    (C) IDENTIFICATION METHOD: computer searching for    sequence similarities.    (x) PUBLICATION INFORMATION:    (A) AUTHORS: Jones, C.L.    Khan, S.A.    (B) TITLE: Nucleotide Sequence of the Enterotoxin B    Gene from Staphylococcus aureus    (C) JOURNAL: J. Bacteriol.    (D) VOLUME: 166    (E) ISSUE: 1    (F) PAGES: 29-33    (G) DATE: APR-1986    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    ThrLysLeuGlyAsnTyrAspAsnValArgValGluPheLysAsnLys    151015    AspLeuAlaAspLysTyrLysAspLysTyrVal    2025    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Staphylococcus aureus    (G) CELL TYPE: unicellular organism    (ix) FEATURE:    (A) NAME/KEY: staphylococcal enterotoxin C1 amino acid    sequence having sequence similarity to    MAM.    (B) LOCATION: residues 30 to 56    (C) IDENTIFICATION METHOD: computer searching for    sequence similarities.    (x) PUBLICATION INFORMATION:    (A) AUTHORS: Bohach, G.A.    Schlievert, P.M.    (B) TITLE: Nucleotide sequence of the staphylococcal    enterotoxin C1 gene and relatedness to    other pyrogenic exotoxins    (C) JOURNAL: Mol. Gen. Genet.    (D) VOLUME: 209    (F) PAGES: 15-20    (G) DATE: 1987    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    AspHisTyrValSerAlaThrLysValLysSerValAspLysPheLeu    151015    AlaHisAspLeuIleTyrAsnIleSerAspLys    2025    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Staphylococcus aureus    (G) CELL TYPE: unicellular organism    (ix) FEATURE:    (A) NAME/KEY: staphylococcal enterotoxin C1 amino acid    sequence having sequence similarity to    MAM.    (B) LOCATION: residues 121 to 147    (C) IDENTIFICATION METHOD: computer searching for    sequence similarities.    (x) PUBLICATION INFORMATION:    (A) AUTHORS: Bohach, G.A.    Schlievert, P.M.    (B) TITLE: Nucleotide sequence of the staphylococcal    enterotoxin C1 gene and relatedness to    other pyrogenic toxins    (C) JOURNAL: Mol. Gen. Genet.    (D) VOLUME: 209    (F) PAGES: 15-20    (G) DATE: 1987    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    AsnHisPheAspAsnGlyAsnLeuGlnAsnValLeuIleArgValTyr    151015    GluAsnLysArgAsnThrIleSerPheGluVal    2025    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Staphylococcus aureus    (G) CELL TYPE: unicellular organism    (ix) FEATURE:    (A) NAME/KEY: staphylococcal enterotoxin C1 amino acid    sequence having sequence similarity to    MAM.    (B) LOCATION: residues 124 to 150    (C) IDENTIFICATION METHOD: computer searching for    sequence similarities.    (x) PUBLICATION INFORMATION:    (A) AUTHORS: Bohach, G.A.    Schlievert, P.M.    (B) TITLE: Nucleotide sequence of the staphylococcal    enterotoxin C1 gene and relatedness to    other pyrogenic exotoxins    (C) JOURNAL: Mol. Gen. Genet.    (D) VOLUME: 209    (F) PAGES: 15-20    (G) DATE: 1987    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    AspAsnGlyAsnLeuGlnAsnValLeuIleArgValTyrGluAsnLys    151015    ArgAsnThrIleSerPheGluValGlnThrAsp    2025    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Streptococcus pyogenes    (G) CELL TYPE: unicellular organism    (ix) FEATURE:    (A) NAME/KEY: streptococcal pyrogenic exotoxin C amino    acid sequence having sequence similarity    to MAM.    (B) LOCATION: residues 109 to 135    (C) IDENTIFICATION METHOD: computer searching for    sequence similarities.    (x) PUBLICATION INFORMATION:    (A) AUTHORS: Goshorn, S.C.    Schlievert, P.M.    (B) TITLE: Nucleotide Sequence of Streptococcal    Pyrogenic Exotoxin Type C    (C) JOURNAL: Infect. Immun.    (D) VOLUME: 56    (E) ISSUE: 9    (F) PAGES: 2518-2520    (G) DATE: SEP-1988    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    SerGlyGluSerGlnGlnAsnLeuAsnAsnLysIleIleLeuGluLys    151015    AspIleValThrPheGlnGluIleAspPheLys    2025    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Mycoplasma arthritidis    (B) STRAIN: PG6    (G) CELL TYPE: unicellular organism    (ix) FEATURE:    (B) LOCATION: MAM residues 16 to 32    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    ValGlnAsnLeuAsnAsnValValPheThrAsnLysGluLeuGluAsp    151015    Ile    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Mycoplasma arthritidis    (B) STRAIN: PG6    (G) CELL TYPE: unicellular organism    (ix) FEATURE:    (B) LOCATION: MAM residues 10 to 35    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    LysAlaGlnLysHisPheValGlnAsnLeuAsnAsnValValPheThr    151015    AsnLysGluLeuGluAspIleTyrAsnLeu    2025    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Murine tumor virus 7    (ix) FEATURE:    (B) LOCATION: MTV7 residues 77 to 104    (x) PUBLICATION INFORMATION:    (A) AUTHORS: Beutner, U.    Frankel, W.N.    Cote, M.S.    Coffin, J.M.    Huber, B.T.    (B) TITLE: Mls-1 is encoded by the long terminal    repeat open reading frame of the mouse    mammary tumor provirus Mtv-7    (C) JOURNAL: Proc. Nat'l Acad. Sci. USA    (D) VOLUME: 89    (F) PAGES: 5432-5436    (G) DATE: JUN-1992    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    SerPheAsnSerSerSerValGlnAspTyrAsnLeuAsnAsnSerGlu    151015    AsnSerThrPheLeuLeuGlyGlnGlyProGlnPro    2025    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Murine tumor virus 7    (ix) FEATURE:    (B) LOCATION: MTV7 residues 115 to 140    (x) PUBLICATION INFORMATION:    (A) AUTHORS: Beutner, U.    Frankel, W.N.    Cote, M.S.    Coffin, J.M.    Huber, B.T.    (B) TITLE: Mls-1 is encoded by the long terminal    repeat open reading frame of the mouse    mammary tumor provirus Mtv-7    (C) JOURNAL: Proc. Nat'l Acad. Sci. USA    (D) VOLUME: 89    (F) PAGES: 5432-5436    (G) DATE: JUN-1992    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    ProSerGluIleGluIleArgMetLeuAlaLysAsnTyrIlePheThr    151015    AsnLysThrAsnProIleGlyArgLeuLeu    2025    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: human immunodeficiency virus-1    (B) STRAIN: 1R    (ix) FEATURE:    (A) NAME/KEY: GAG protein    (B) LOCATION: residues 22 to 47    (x) PUBLICATION INFORMATION:    (A) AUTHORS: Starcich, B.R.    Hahn, B.H.    Shaw, G.M.    McNeely, P.D.    Modrow, S.    Wolf, H.    Parks, E.S.    Parks, W.P.    Josephs, S.F.    Gallo, R.C.    Wong-Staal, F.    (B) TITLE: Identification and characterization of    conserved and variable regions in the    envelope gene of HTLV-III/LAV, the    retrovirus of AIDS.    (C) JOURNAL: Cell    (D) VOLUME: 45    (F) PAGES: 637-648    (G) DATE: 1986    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    ArgProArgGlyLysLysArgTyrLysLeuLysHisIleValTrpAla    151015    SerArgGluLeuGluArgPheAlaValAsn    2025    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: human immunodeficiency virus-1    (B) STRAIN: 1R    (ix) FEATURE:    (A) NAME/KEY: GAG protein    (B) LOCATION: residues 129 to 154    (x) PUBLICATION INFORMATION:    (A) AUTHORS: Starcich, B.R.    Hahn, B.H.    Shaw, G.M.    McNeely, P.D.    Modrow, S.    Wolf, H.    Parks, E.S.    Parks, W.P.    Josephs, S.F.    Gallo, R.C.    Wong-Staal, F.    (B) TITLE: Identification and characterization of    conserved and variable regions in the    envelope gene of HTLV-III/LAV, the    retrovirus of AIDS.    (C) JOURNAL: Cell    (D) VOLUME: 45    (F) PAGES: 637-648    (G) DATE: 1986    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    SerGlnAsnTyrProIleValGlnAsnLeuGlnGlyGlnMetValHis    151015    GlnAlaIleSerProArgThrLeuAsnAla    2025    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: human immunodeficiency virus-1    (B) STRAIN: Z321    (ix) FEATURE:    (A) NAME/KEY: envelope protein    (B) LOCATION: residues 543 to 568    (x) PUBLICATION INFORMATION:    (A) AUTHORS: Srinivasan, A.    York, D.    Butler, D., Jr.    Jannoun- Nasr, R.    Getchell, J.    McCormick, J.    Ou, C.- Y.    Myers, G.    Smith, T.    Chen, E.    Flaggs, G.;Berman, P.;Schochetman, G.;Kalyanaramen, S.    (B) TITLE: Molecular characterization of HIV-1    isolated from a serum collected in 1976:    nucleotide sequence comparison to recent    isolates and generation of hybrid HIV.    (C) JOURNAL: AIDS Res. Hum. Retroviruses    (D) VOLUME: 5    (F) PAGES: 121-129    (G) DATE: 1989    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    ArgLeuLeuSerGlyIleValGlnGlnGlnAsnAsnLeuLeuArgAla    151015    IleGluAlaGlnGlnHisLeuLeuLysLeu    2025    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: human immunodeficiency virus-1    (B) STRAIN: Z321    (ix) FEATURE:    (A) NAME/KEY: envelope protein    (B) LOCATION: residues 600 to 625    (x) PUBLICATION INFORMATION:    (A) AUTHORS: Srinivasan, A.    York, D.    Butler, D., Jr.    Jannoun- Nasr, R.    Getchell, J.    McCormick, J.    Ou, C.- Y.    Myers, G.    Smith, T.    Chen, E.    Flaggs, G.;Berman, P.;Schochetman, G.;Kalyanaramen, S.    (B) TITLE: Molecular characterization of HIV-1    isolated from a serum collected in 1976:    nucleotide sequence comparison to recent    isolates and generation of hybrid HIV.    (C) JOURNAL: AIDS Res. Hum. Retroviruses    (D) VOLUME: 5    (F) PAGES: 121-129    (G) DATE: 1989    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    GlyLysIleIleCysProThrAsnValProTrpAsnSerSerTrpSer    151015    AsnLysSerGlnSerAspIleTrpAspLys    2025    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: human immunodeficiency virus-1    (B) STRAIN: Z321    (ix) FEATURE:    (A) NAME/KEY: envelope protein    (B) LOCATION: residues 629 to 654    (x) PUBLICATION INFORMATION:    (A) AUTHORS: Srinivasan, A.    York, D.    Butler, D., Jr.    Jannoun- Nasr, R.    Getchell, J.    McCormick, J.    Ou, C.- Y.    Myers, G.    Smith, T.    Chen, E.    Flaggs, G.;Berman, P.;Schochetman, G.;Kalyanaramen, S.    (B) TITLE: Molecular characterization of HIV-1    isolated from a serum collected in 1976:    nucleotide sequence comparison to recent    isolates and generation of hybrid HIV.    (C) JOURNAL: AIDS Res. Hum. Retroviruses    (D) VOLUME: 5    (F) PAGES: 121-129    (G) DATE: 1989    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    LeuGluTrpAspLysGluValSerAsnTyrThrGlnValIleTyrAsn    151015    LeuIleGluGluSerGlnThrGlnGlnGlu    2025    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Human Immunodeficiency Virus    (ix) FEATURE:    (A) NAME/KEY: internal region of GAG protein    (B) LOCATION: 135 to 142    (D) OTHER INFORMATION: contains conservative sequence    homology with MAM amino acids 16-23    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    ValGlnAsnLeuGlnGlyGlnMet    15    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Human Immunodeficiency Virus    (B) STRAIN: Z321    (ix) FEATURE:    (A) NAME/KEY: internal region of envelope protein    (B) LOCATION: 549 to 556    (D) OTHER INFORMATION: contains conservative sequence    homology with MAM amino acids 16-23    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    ValGlnGlnGlnAsnAsnLeuLeu    15    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Murine Tumor Virus 7    (ix) FEATURE:    (B) LOCATION: 83 to 90    (D) OTHER INFORMATION: contains conservative sequence    homology with MAM amino acids 16-23    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    ValGlnAspTyrAsnLeuAsnAsn    15    __________________________________________________________________________

We claim:
 1. A purified polynucleotide having a nucleotide sequence ofSEQ ID NO:3.